A5 QUANTIFICATION OF TRANSPORTER EXPRESSION IN THE PLASMA MEMBRANE VS. INTRACELLULAR COMPARTMENTS USING BIOTINYLATION AND LC-MS/MS BASED PROTEOMICS

Vineet Kumar , Pharmaceutics, University of Washington, Seattle, WA
Bhagwat Prasad , Pharmaceutics, University of Washington, Seattle, WA
Jashvant D. Unadkat , Pharmaceutics, University of Washington, Seattle, WA
Purpose: We and others have proposed that in vivo transporter-based hepatobiliary clearance can be predicted by scaling in vitro intrinsic transporter clearance determined in cells transfected with the relevant transporters.  This scaling can be achieved by quantifying the expression of transporters in the cell line vs. that in liver tissue. Quantitative proteomics using LC-MS/MS has become a powerful tool to quantify the expression of transporters in cells and tissues.  However, this method, as currently used, quantifies the expression of transporters in the total cell membranes and not just in the plasma membrane. Here we report data on optimization of a biotinylation protocol for isolation of pure plasma membranes from transporter expressing cell lines. The expression of a transporter in the plasma membrane vs. intracellular compartments was quantified using LC-MS/MS based proteomics. 

Methods: The Pierce cell surface isolation kit was used (1). OATP1B1 overexpressing CHO cells were incubated with various concentrations of the membrane impermeable biotinylation reagent, sulfosuccinimidyl-2-[biotinamido]ethyl-1,3-dithiopropionate (EZ-Link Sulfo-NHS-SS-Biotin), for 1 hr at 4°C (0.24 mg/mL, 0.78 mg/mL, 1.02 mg/mL, 1.26 mg/mL or 1.5 mg/mL) or 37°C (0.24 mg/mL, 0.78 mg/mL or 1.5 mg/mL).  Biotinylation reaction was quenched and the cells were treated with the lysis buffer. The lysate was incubated at room temperature for 1 hr with a column containing NeutrAvidin resin. After incubation, the column containing NeutrAvidin resin was centrifuged (1000 xg for 1 min) and the non-biotinylated fraction (containing intracellular proteins) was collected.  Then, the column containing NeutrAvidin resin was incubated with the elution buffer containing dithiothreitol to elute the biotinylated fraction (containing the plasma membrane proteins). The expression of OATP1B1, Na+/K+-ATPase (plasma membrane marker), GAPDH (cytosol marker) and calreticulin (endoplasmic reticulum marker) was quantified in the lysate, the non-biotinylated and the biotinylated fractions using LC-MS/MS (2).

Results: Both the concentration of the biotinylation reagent and the incubation temperature significantly affected the extent of biotinylation of plasma membrane proteins. For complete biotinylation of plasma membrane proteins, incubation of cells with 0.78 mg/mL of sulfo-NHS-SS-biotin at 37°C was necessary. Using these conditions, <14% of GAPDH or calreticulin was biotinylated indicating purity of the plasma membrane isolated as well as lack of intracellular penetration of the biotinylation reagent. With these optimal conditions, OATP1B1 was observed to be almost completely (94%) expressed in the plasma membrane of CHO-OATP1B1 cells with very little presence in the intracellular compartments.

Conclusions: OATP1B1 transporter is predominately expressed in the plasma membrane of CHO-OATP1B1 cells. The optimized biotinylation conditions can now be used to determine the plasma membrane expression of other transporters expressed in either cell lines or human hepatocytes to arrive at scaling factors for IVIVE of transporter-based disposition of drugs.

Acknowledgement: This work was supported in part by the Simcyp Grant & Partnership Scheme 2013/2014.

Reference:

  1. https://tools.lifetechnologies.com/content/sfs/manuals/MAN0011183_EZ_SulfoNHS_SS_Biotin_UG.pdf

  2. Kumar V, Prasad B, Patilea G, Gupta A, Salphati L, Evers R, Hop CE, Unadkat JD (2015) Quantitative transporter proteomics by liquid chromatography with tandem mass spectrometry: addressing methodologic issues of plasma membrane isolation and expression-activity relationship. Drug Metab Dispos 43(2):284-288